Currently there is no potent multivalent vaccine against Venezuelan Equine Encephalitis (VEE) that can protect research laboratory workers or provide a biowarfare countermeasure. Several attenuated VEE vaccines have been tested, including a strain called TC-83, which is currently licensed for use in horses. However, these vaccines are too dangerous for human use or face a difficult regulatory approval process because of neurotropism effects. Previously published studies demonstrate that a recombinant adenovirus expressing E2, E3 and 6K genes (E2E36K) of TC-83 can safely protect against aerosolized VEE in animal models. Unfortunately, the large amount of recombinant adenovirus necessary to provide protection is impractical for producing a human vaccine. Preliminary data in this proposal demonstrate that West Coast Biological's (WCB) patent pending oral vaccine platform, which uses an expressed Toll Receptor 3 ligand in conjunction with antigen expressed from a non-replicating adenovirus vector, can significantly augment the adaptive immune response. We propose to develop a safe and potent oral vaccine vector that is effective against multiple serotypes of VEE, and produce it as a room temperature stable vaccine tablet. Aim 1 of this proposal investigates whether WCB's adjuvanted recombinant adenovirus vector in combination with VEE antigen, can elicit a protective antibody response. In aims 2 and 3, we will test an antigen from the Mucambo strain to determine if it provides protection against epizootic forms of VEE, and use this information to construct a broadly protective VEE vaccine vector. Aim 4 focuses on developing a room temperature stable form of the vaccine. In Aim 5, we will use GMP material in toxicology tests to demonstrate the safety of WCB's VEE vaccine. We will evaluate the potency of the new vaccines by measuring antibody responses and performing challenge studies in mice. Together, these aims will map a development plan to commercialize a VEE vaccine as well as the vaccine platform. Public Health Relevance: West Coast Biologicals (WCB) is developing a new vaccine to prevent infection by Venezuelan Equine Encephalitis (VEE), a pathogen that can infect laboratory workers and has biowarfare potential. Because of the dangers involved with previous vaccines, we propose to develop a safer vaccine that is effective against multiple forms of the virus, and formulate it as a room-temperature stable tablet.